The invention relates to the use of nonradioactive tracer technology used to measure DNA synthesis rates. DNA synthesis is determined by administering one or more stable isotope labelled precursors to one or more of DNA substituents and the amount of the stable isotope label in the DNA that is extracted from cells that are of interest is measured.
The chemical reaction interface for mass spectrometry (CRIMS) was first developed by Markey and Abramson in 1982 as a way of utilizing the mass spectrometer as an element (carbon) and isotope (14C) selective detector for gas chromatography. Chace and Abramson further developed the technique to encompass a range of stable isotopes and were the first to show the application of this technique to drug metabolism. The principle of CRIMS is for the chromatographic effluent to flow into a microwave-powered reaction cell where the molecular species are broken down to elements. The continuous addition of a reactant gas to the microwave-induced plasma results in the formation of simple gaseous reaction products that are characteristic of the elements contained in the original molecule. Conventional electron ionization mass spectrometry is then used to detect elements or isotopes in these reaction products. Chemical Reaction Interface/Mass Spectrometry {CRIMS} has evolved from a concept into a selective, sensitive, and versatile technique by which targeted isotopes or elements can be monitored in studies of metabolism. CRIMS parallels the use of radioisotopes in that each technique monitors for its tracers independent from the chemical structures in which the targeted species exist. Using CRIMS, intact analyses are decomposed to their elemental species in a high temperature electronic plasma and interact with atoms of a reactant gas to form a set of new, small polyatomic species that are detected by a conventional mass spectrometer. The presence of a given polyatomic species signifies the presence of a particular element, the abundance of that species quantifies it, and the isotopic signature of that species can differentiate it from endogenous materials. By subtracting the known natural abundance of the stable isotope that is used as a tracer, a chromatogram showing only enriched species can be produced. CRIMS has been interfaced with gas and liquid chromatography, and a number of applications have been carried out.
Some isotope metabolic assays are known. For example, U.S. Pat. No. 4,182,656 discloses a method for detecting the presence of biologically active agents, utilizing a 13C-labeled substrate. The method uses 13C-labeled glucose, allows fermentation to take place and then measures the proportions of 13C and 12CO2 in a culture gas to ascertain whether there has been increase in the ratio of the 13C16O2 indicative of the presence of a biological active agent. Col. 10, line 45 indicates that the gas is run through a mass spectrum to determine the reference value for the ratio of CO2.
U.S. Pat. No. 4,830,010, discloses methods for the diagnosis of gastrointestinal disorders. The method involves using amounts of isotope-labeled urea. The urea is labeled with one or more stable isotopes. The individual""s breath is tested for the presence of isotope-labeled carbon dioxide or isotope-labeled ammonia which are hydrolysis products of campylobacter pyloridis. The gas sample is tested by mass spectroscopy.
U.S. Pat. No. 5,559,038 discloses a method for quantifying the amounts of oxidized sulfhydryl amino acids particularly cysteine sulfinic acid and cysteic acid in biological samples. The oxidized sulfhydryl amino acids are measured by gas chromatography/mass spectrometry after derivatization, using stable isotope internal standards. Deviation from normal levels indicates neuropsychiatric disorders.
Chemical Abstracts, Vol. 95, No. 7, Abstract No. 62629g discloses the xe2x80x9cLongitudinal relaxation in a homonuclear coupled two-spin system. The case of (13C, 2H) glycinexe2x80x9d. The abstract discusses a synthesis of amino acids and peptides. J. Magn. Reson, 1981, Vol 42.
Chemical Abstracts, Vol. 90, No. 17, Abstract No. 13595Op discloses a xe2x80x9cStudy of the effects of truncal subdiaphragmatic vagotomy and pyloroplasty on the nitrogen balance in bionomic gastric surgery in rats using the stable isotope-labelled amino acid 15N-glycine.
Chemical Abstracts, Vol. 89, No. 17, Abstract No. 147218y discloses the synthesis of cyclic peptides from glycine partially labeled with deuterium and/or carbon-13 and two pairs of proline.
Chemical Abstracts, Vol. 111, No. 13, Abstract No. 111948v discloses stable isotope dilution analysis of n-hexanoylglycine, 3-phenylpropionylglycine and suberylglycine in human urine using chemical ionization gas chromatography/mass spectrometry selected ion monitoring. Biomed. Environ. Mass Spectrom, 1989 VOLUME: 18 NUMBER: 7, p 471-7.
Chemical Abstracts, Vol. 111, No. 11, Abstract No. 94305p discloses the xe2x80x9cMeasurement of apolipoprotein B synthesis in perfused rat liver using stable isotopes such as 15N hippurate as a measurement of the intracellular 15N glycine precursor enrichmentxe2x80x9d. The isotopes are used in measurement of ribonucleic acids, and are glycine specific. See J. Lipid. Res., Vol. 30, No. 6 (1989) p. 841-6.
Chemical Abstracts, Vol. 87, No. 1, Abstract No. 1970z discloses xe2x80x9cA stable isotope method for measurement of thymidine incorporation in DNA. The abstract discloses the isotope is a cancer therapy cell growth stable isotope. Heck, Henry d""A.; McReynolds, James H.; Anbar, Michael; Cell tissue Kinet. 1977, Vol 10, NUMBER: 2, p. 111-19. Heck et al. use a stable isotope labeled precursor to DNA and measuring the increased DNA labeling. They did not use any de novo precursors, such as the glycine. They do not do any separations on-line. They separate each nucleic acid with thin-layer chromatography and take each purified spot for MS analysis.
Song and Abramson, Nitrogen Trifluoride: A New Reactant gas in chemical reaction interface mass spectrometry for detection of phosphorus, Deuterium, Chlorine and Sulfur. J. Am. Soc. Mass Spectrom., No. 6, 1995, pp. 421-427. This publication discloses generally, that chemical reaction interface mass spectrometry (CRIMS) may be used for studying metabolism without the use of radioactive labels.
Slatkin et al., Stable Isotopes, Proceedings of the Third International conference. A Postmortem study of stable carbon Isotope ratios in human Cerebellar DNA: Preliminary Results. This study showed that the stable carbon isotope ratios in human cerebellar DNA show different levels in Europeans and Americans/Canadians. DNA was isolated combusted, and purified CO2 on a gas chromatograph, and measured with a mass spectrometer. This work did not attempt to alter the isotope ratio of DNA by giving a labeled precursor and were examining how different diets in different cultures lead to different 13C ratios in subjects"" DNA. Similar measurements have been made for other parts of the body, e.g., proteins, egg shell carbonate, etc.
Berthold et al., Evidence for incorporation of intact dietary pyrimidine (but not purine) nucleosides into hepatic RNA, Proc. Nat. Acad. Sci., Vol 92, page 10123-10127. This publication reports the growth of spirulina algae in 13C labelled environment. The spirulina algae is fed to mice. Hepatic analysis of these animals showed large quantities of dietary pyrimidines are incorporated into hepatic nucleic acids. Almost no labelled purine nucleosides are incorporated into hepatic ribonucleic acids (RNA). Testing was performed using gas chromatography/mass spectrometry.
Strong et al., J. Biolog. Chem., Vol. 260 No. 7, Apr. 10, 1985, pp. 4276-4281. A Novel Approach to the Analysis of Mass Spectrally Assayed Isotope-labelling Experiments. This publication discloses the use of 15NH4Cl and L-glutamine as precursors in the study of de novo pyrimidine (uracil) biosynthetic pathways. Quantitation of the labelled uracil was performed with a mass spectrometer. The authors were not examining DNA synthesis rates.
Macallan, et al present a different approach to measuring DNA synthesis rates with stable isotopes and mass spectrometry. Macallan, et al has shown that DNA synthesis rates can be measured with a stable isotopic precursor and mass spectrometry. Their method differs markedly from the inventor""s method in a number of substantive ways. The precursor, [6,6-2H2]-glucose, is not incorporated into the nucleic acid base as does the present method, but labels the deoxyribose component. As such, every component of DNA will be labeled, thus failing to provide any internal standards. Furthermore, glucose is a nutrient with high blood concentrations that requires at least 100-fold more labeled material to achieve a comparable isotopic enrichment compared to glycine or any of the nucleosides or bases that comprise DNA. A second major difference is that the method of Macallan uses gas chromatography (GC) to separate and introduce the DNA components rather than the high performance liquid chromatography (HPLC) that the inventor has used. With GC the nucleosides have to be chemically derivatized, a process that contributes to a 16% interference at the mass where the labeled DNA-derived materials are detected. With the preferred use of HPLC, this interference is reduced because no derivatization is required. A third difference is that Macallan examines the intact derivatized nucleosides using mass spectrometry while the inventor has used CRIMS. By doing so, the invention has only a 1% interference at the mass where the labeled DNA-derived materials are detected. Finally, by using an IRMS rather than the conventional MS approach of Macallan, the inventor is able to determine perhaps 1000-fold lower isotopic enrichments, thus making that approach much more practical. Macallan examined cells in culture, intestinal epithelium, thymus, and liver cells from rats, and granulocytes from humans.
The current state of the art uses a radiolabelled tritiated thymidine for metabolic diagnostic testing. Thus, there is a need in the art for sensitive DNA synthesis testing and diagnostic testing without the use of a radiolabel. The present invention overcomes deficiencies in the prior art and provides for a sensitive assay to provide measurement of DNA synthesis without the use of a radioactive label.
In a preferred embodiment the invention provides an assay for detection of DNA synthesis comprising the steps of
(a) adding a stable isotope labelled DNA precursor to a replicating DNA sample;
(b) isolating a portion of said replicating DNA sample comprising said stable isotope; and
(c) measuring the enrichment of stable isotope label in newly synthesized DNA with an isotope ratio mass spectrometer, wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
In an alternative embodiment the invention provides a method of measuring the enrichment of stable isotopes of hydrogen, carbon, oxygen and nitrogen in replicating DNA comprising the steps of:
(a) adding a stable isotope labelled DNA precursor to a replicating DNA sample;
(b) isolating a portion of said replicating DNA sample comprising said stable isotope; and
(c) measuring the enrichment of stable isotope label in newly synthesized DNA with a chemical reaction interface mass spectrometer, wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
In still another embodiment the invention provides an assay for detection of DNA synthesis comprising the steps of
(a) adding a stable isotope labelled DNA precursor to a replicating DNA sample;
(b) isolating or purifying a portion of said replicating DNA sample comprising said stable isotope;
(c) combusting the DNA of step (b) to produce a stable isotope labelled combustion product, (e.g. CO2) on which an enrichment measurement is made;
(d) measuring the enrichment of stable isotope label in newly synthesized DNA with a mass spectrometer, wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
Advantageously the invention provides an assay for detection of DNA synthesis comprising the steps of
(a) adding a stable isotope labelled precursor to a replicating DNA sample;
(b) isolating or purifying a portion of said replicating DNA sample comprising said stable isotope;
(c) optionally degrading the DNA of step (b);
(d) analyzing the DNA obtained in steps (b) or (c) by chromatography; and
(e) measuring the enrichment of stable isotope label in newly synthesized DNA with a mass spectrometer, wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
Still another embodiment provides an assay for detection of DNA synthesis comprising the steps of
(a) adding a stable isotope labelled DNA precursor to DNA, wherein said labelled DNA precursor does not label all four DNA bases;
(b) isolating or purifying a portion of said replicating DNA sample comprising said stable isotope;
(c) degrading the DNA of step (b);
(d) analyzing the degraded DNA by high performance liquid chromatography, wherein said chromatography allows the non-labeled DNA components to serve as an internal standard for the labeled DNA; and
(e) measuring the amount of stable isotope label in the DNA by chemical reaction interface mass spectroscopy (CRIMS); wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
An application of the assay of the invention provides a method of measuring the effectiveness of chemotherapeutic drugs comprising
(a) administering a stable isotope labelled precursor to DNA to a patient who has received chemotherapy, wherein said labelled precursor does not label all four DNA bases;
(b) obtaining a replicating DNA sample from said patient who has received chemotherapy;
(c) isolating or purifying a portion of said replicating DNA sample comprising said stable isotope;
(d) degrading the DNA of step (c);
(e) analyzing the degraded DNA by chromatography (preferably high performance liquid chromatography), wherein said chromatography allows the non-labeled DNA components to serve as an internal standard for the labeled DNA; and
(f) measuring the amount of stable isotope label in the DNA by mass spectroscopy;
wherein the stable isotope labelled precursor to DNA is incorporated via de novo purine or pyrimidine biosynthesis or by biochemical metabolic salvage pathways and is synthesized into a new strand of DNA.
The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.